Project Summary O-glycosylation of nuclear and cytoplasmic proteins by a single ?-N-acetyl-D-glucosamine moiety (O- GlcNAc) is a common post-translational modification that is highly dynamic and fluctuates in response to cellular stimuli. This type of glycosylation has been found on approximately a thousand human proteins to date, and is thought to be nearly as wide-spread and abundant as protein phosphorylation. In fact, O-GlcNAc often competes with protein phosphorylation, and these two modifications have extensive crosstalk in the regulation of signaling, transcription, and the functions of oncogenes and tumor suppressors. The modification appears to play a major role in key pathophysiological conditions including cancer, Alzheimer?s disease, and diabetes. Many of the first proteins identified carrying this modification were transcription factors, and it has become clear in the last several years that O-GlcNAc plays a major role in chromatin remodeling and gene expression. The focus of this proposal is to develop site-specific antibodies that can be used as tools in the elucidation of the role that O-GlcNAc plays in epigenetics. In the predecessor Phase I grant we focused on evaluating synthetic immunogens, the production of polyclonal antibodies (PAbs) to five sites of O-GlcNAc modification on the four core histones (Histone 2A, 2B, 3, and 4), and characterizing these antibodies. We were successful with each Aim of this previous project, which leads to this Phase II proposal. Here, we propose to utilize our proprietary immunization strategy to significantly expand our repertoire of site-specific O-GlcNAc antibodies to include the majority of proteins currently known to be modified in this manner that are also involved in gene expression. Consequently, if we are successful, researchers will have access to a wide-range of site-specific Abs developed for epigenetic research, and thus we feel that this study will have an immediate impact on epigenetic research and could have far reaching implications in disease research.